The subject matter of the invention is a tissue adhesive as well as stabilized protein preparations for a tissue adhesive or preparations for parenteral application, which have no significant loss of effect after being stored in a liquid state or after being defrosted following storage in a frozen state and during further storage in a liquid state over a period of several months or years.
It is already known that tissue adhesives can be used to connect human or animal tissue, and that the main components of such tissue adhesives include fibrinogen, Factor XIII and thrombin. These protein preparations require careful stabilization to retain their full effect and their application properties until they are used for gluing tissue during surgery.
Tissue gluing is a method that has already been described at the turn of the century and repeatedly thereafter (S. Bergel: On the effects of fibrin. Dt. med. Wschr. 35:663-5 (1909); E. P. Cronkite, E. L. Lozner, J. M. Deaver: Use of thrombin and fibrinogen in skin grafting. JAMA 124:976-8 (1944); H. Matras; H. P. Dinges, H. Lassmann, B. Mammoli, Wr. Med. Wschr. 37:517 (1972); H. Matras, H. P. Dinges, H. Lassmann, B. Mammoli: J. max. fac. Surg. 1:37 (1973); H. Matras, F. Braun, H. Lassmann, H. P. Ammrer, B. Mammoli: Plasma clot welding of nerves (experimental report), J. max. fac. Surg. 1:2364 (1973); H. Kudema, H. Matras, Wiener klin. Wschr. 87:495-496 (1975). Initially, plasma or fibrinogen was still used as powder; later on, purified fibrinogen was used, for example, in the form of a cryoprecipitate.
With the commercial production of fibrinogen-/Factor XIII- and thrombin concentrates since the Seventies, the importance of tissue gluing has increased significantly. It is now used, for example, to support sutures, local haemostasis, the sealing of body cavities to avoid loss of fluid, and for wound care. Tissue gluing with fibrin adhesives is a physiological method and therefore has advantages compared to the synthetic adhesives with respect to compatibility and biodegradability of the adhesive components.
Tissue adhesives are commercially available either as lyophilizates or as frozen preparations. However, after reconstitution or after defrosting, the products are stable in solution for only a few days, because with the highly concentrated fibrinogen-/Factor XIII-solutions, an aggregation and therefore an (for example, proteolytic) inactivation occurs, which makes any further use impossible.
The tissue adhesives that have been described in the literature until now are not yet commercially available and are generally comprised of frozen or freeze-dried components that must be defrosted or dissolved prior to use. To improve the processing, the solubility, the defrosting or the stability of the fibrinogen concentrate, European patent 0,085,923, German patent application 196,17,369 and European patent application 0,856,317 describe the use of chaotropic agents or additives such as arginine or urea or their derivatives or derivatives of benzene, imidazole, pyrozol, furan, thiazole and purine, which generally improve the solubility of proteins. Chaotropic agents in this context are agents that reduce or destabilize the reciprocal effect between proteins or parts thereof and therefore reduce their tendency towards aggregation. It is important to guarantee the stability of the components such as fibrinogen and Factor XIII even in the presence of said chaotropic agents and under the selected conditions. So far, this has not been successful with frozen fibrinogen-/Factor XIII-concentrations that had to be stored for several weeks or months in liquid form after defrosting, or with fibrinogen-/Factor XIII-concentrations that could be stored only in liquid form.
With liquid storage, but especially also with storage in frozen state, the loss of F XIII-activity in the formulations described thus far was so high that in the presence of effective quantities of chaotropic agents, the F XIII-content often clearly drops after only a few weeks or months, often even below the detection limit.
With the formulations in accordance with European patent application 0,856,317, it was shown that tranexaminic acid (AMCA), especially in the presence of chaotropic agents such as arginine and inorganic salts, clearly reduced the F XIII content in the course of storage at xe2x88x9220xc2x0 (Table 1 b, Batch 1). Storage at 4xc2x0 Celsius leads to an increase in viscosity in this formulation (Table 1 a, Batch 1), which also rules out a long-term term storage. Thus, these formulations must be considered non-stable in view of the simultaneous stability of fibrinogen and F XIII. Formulations in accordance with DE 196,17,369 also indicate problems in maintaining F XIII-activity (see Table 1, Batch 2 and 2).
Another biological adhesive for human or animal tissue is known from the European patent specification 0,487,713. Said adhesive is stabilized in liquid form at low temperatures. This is supposed to be achieved in that the preparation containing fibrinogen comprises at least one chaotropic agent in a concentration between approximately 0.3 M and 1 M and in that the adhesive is liquid at the storage temperature.
Such fibrinogen concentrate typically comprised about 4 mmol tri-sodium citrate, 240 mmol NaCl, 80 mmolxe2x80x94amino caproic acid (EACA), 240 mmol glycine, 1% polysorbate, 0.6 grams/liter sodium caprolate, 0.5 mol urea, 2,000 KIE/ml aprotinin, if necessary, and a pH of 7.5. The stability was evaluated after only one month, which is very short for a therapeutic preparation. The F XIII-activity was not analyzed. J. Chabbat et al. reported about a fibrinogen concentrate that remains stable in liquid state at 4xc2x0 Celsius over a period of six months (J. Chabbat, M. Tellier, P. Porte and M. Steinbuch: Properties of a new fibrin glue stable in liquid state. Thromb. Res. 76: 525-533 (1994)). In addition to other formulation components, typically 60 mmol/liter NaCl, 20 mmol/liter EACA and 60 mmol/liter glycine, this concentration comprised 0.5 mol urea or 5% arginine (=0.29 mol). However, the F XIII-strength of this concentration was also not tested.
These liquid formulations, which were described in the European patent specification 0,487,713 and in the literature, are characterized in that the aggregation (polymerization) and thus the increase in viscosity of the concentrated fibrinogen component, is prevented or reduced at refrigeration temperatures. However, Factor XIII, an essential component of fibrinogen concentrates for fibrin glues, is inactivated to a greater or lesser degree under these conditions. In the formulations provided for storage in cooled state in accordance with European patent specification 0,487,713 or the related publication by Chabbat et al. (J. Chabbat, M. Tellier, P. Porte and M. Steinbuch: Properties of a new fibrin glue stable in liquid state. Thromb. Res. 76:525-533 (1994)), the instability of F XIII is therefore a significant problem that is not solved by the proposed formulations (see Table 1, Batches 4-5). Furthermore, the strength of chaotropic agents is relatively high at 0.3 to 1.0 mol/liter, which makes lesser concentrations of chaotropic agents appear desirable ( less than 0.3 mol/liter).
Thus, it can be noted that it was found in the analysis of the stability of fibrinogen/Factor XIII preparations as well as the viscosity of various known fibrinogen/Factor XIII-preparations in refrigerated state (0 to 10xc2x0 Celsius) or frozen state with subsequent storage in refrigerated state (0 to 10xc2x0 Celsius) that the previously described formulations do not lead to stable protein preparations. Either the fibrinogen or Factor XIII show a significant reduction in activity during the storage time, or the aggregation of fibrinogen leads to a viscous material that can no longer be applied (see Table 1, Batches 1 to 5).
Thus, the problem to be solved was to develop protein preparations that are liquid and stable over several months, or frozen and stable over several months following defrosting, in which the fibrinogen and/or Factor XIII are stabilized over months or years without any significant loss of effect.
The problem is solved with stabilized protein preparations that in comparison to the state of the art have the advantage that in a first embodiment not only fibrinogen but also Factor XIII is stabilized by the additives and that the content of chaotropic reactants can be reduced, or that in a second embodiment fibrinogen and Factor XIII are formulated separately and thus remain stable.
This is achieved in that for frozen preparations and preparations that must be kept stable for several weeks or months following defrosting, a chaotropic agent corresponding to the definition provided here is used in a lower concentration to avoid the aggregation of fibrinogen, and that the concentration of inorganic salts is reduced and that, if necessary, an antifibrinolytic as well as other common additives and buffer substances are used. A fibrinogen preparation used for this purpose can also contain Factor XIII from the starter material as well as other plasma proteins, such as fibronectin and von Willebrand-Factor (vWF), or it can contain purified Factor XIII as an additive.
Aprotinin or lysine or e-amino caproic acid (EACA) or p-aminomethylbenzoic acid (PAMBA) or their physiologically safe salts can be used as an antifibrinolytic. Studies on the influence of various antifibrinolytics have surprisingly shown that lysine, PAMBA or EACA do not have a negative effect on the activity of F XIII, while tranexaminic acid does. Especially with frozen fibrinogen/F XIII mixtures, but also with fibrinogen/F XIII mixtures stored in liquid state, the use of EACA or lysine is therefore preferred to the use of AMCA. Other stabilizers can be used for F XIII, such as sodium citrate, amino acids and sugar.
Instead of the aforementioned protein preparations, which comprise Factor XIII as well as fibrinogen and their respective stabilizers, it is also possible, and for reasons of better stability even preferable, to store both concentrations separately and only mix them with the thrombin-containing preparation immediately prior to using them as tissue adhesive. The subject matter of the invention is therefore also a tissue adhesive that is comprised of a solution that contains the stabilized factor XIII, a solution that contains the stabilized fibrinogen, and a solution that contains stabilized thrombin, which are provided separately in one packaging unit prepared to be used together. Another advantage of this is that the ratio of Factor XIII and fibrinogen can be changed and adapted to the specific situation as needed.
A) Frozen or lyophilized concentrates that can be stored in liquid state for several weeks/months at 0 to 10xc2x0 Celsius (see Table 1)
Stable, frozen fibrinogen concentrates are known and have been described, but their stability after defrosting is limited to a few days. The limited stability of the fibrinogen concentrate is, among other things, also attributable to the fact that the viscosity soon increases due to the aggregation of the fibrinogen. It is possible to obtain a low viscosity in the liquid state by adding compounds that prevent aggregation, i.e., chaotropic compounds, but these agents have the disadvantage that they lead to a drop in the Factor XIII-activity in frozen state (for example, at xe2x88x9220xc2x0 Celsius). Generally, the loss of F XIII-activity occurs in proportion to the concentration of chaotropic agents, i.e., the higher the concentration of chaotropic agents, the quicker the loss of F XIII-activity.
In the development of the stabilized protein preparations in accordance with the invention, it was now found that not all chaotropic agents have the same influence on the stability of the Factor XIII, and that the other additives to be added in accordance with the invention also have a significant influence on the Factor XIII stability and on the viscosity affected by the fibrinogen aggregation. For example, at the same molarity, arginine is significantly more effective in the prevention of fibrinogen polymerization or aggregation than urea. Furthermore, anti-fibrinolytic additives such as the xcex5-amino caproic acid (EACA), p-aminomethylcyclohexanecarboxylic acid (AMCA) or p-aminomethylbenzoic acid (PAMBA) as well as their physiologically safe salts have an effect on fibrinogen aggregation and F XIII stability. AMCA, especially, has a negative effect on F XIII-activity at storage in frozen state. Surprisingly, EACA, which has a chemical structure very similar to that of AMCA, does not cause the same Factor XIII-drop as AMCA under appropriate conditions. It was further found that F XIII-activity in frozen protein concentrates is not reduced in the presence of specific concentrations of chaotropic agents when the addition of inorganic salts, which until now was common in preparations of this type, is completely abandoned or limited as much as possible. Thus, in the preparation developed in accordance with the invention, fibrinogen and Factor XIII remain liquid and the activity is maintained for at least several weeks or even months after freezing and defrosting, if said formulation comprises a chaotropic compound in a quantity of less than 0.28 mol/liter of a substance that avoids or reduces the aggregation of fibrinogen. Arginine in a quantity of approximately 2 percent by weight has proven especially advantageous. Other slightly chaotropic agents such as citrulline, nicotine amide, urea, etc. or mixtures thereof, for example with arginine, can be used in a quantity of up to 0.28 M, especially of 0.1 to 0.20 M. Furthermore, it is also possible to add water-soluble inorganic salts in concentrations of xe2x89xa6100 mmol/liter, especially xe2x89xa650 mmol/liter, and in particular, preferably in concentrations of xe2x89xa620 mmol/liter in addition to the anti-fibrinolytic compound.
In one of the preparations in accordance with the invention, the fibrinogen as well as Factor XIII remain stable for at least several weeks or months during storage in frozen as well as in liquid state. The addition of other components, such as salts of citric acid or lactic acid or one or several amino acids or a mono- or disaccharide or a sugar alcohol or one of their mixtures can also favorably influence the stability. With these compositions, the preparation in accordance with the invention can be refrozen and defrosted or refrozen after reconstitution of a fibrin glue lyophilizate and stored in frozen condition as a stable fibrinogen/F XIII preparation. This is a further advantage of the formulations in accordance with the invention because refreezing is not possible with the commercial frozen or lyophilized protein preparations that are used as tissue adhesives. This property simplifies the handling of lyophilizates after the reconstitution, or of frozen stored preparations if the entire quantity is not used in one process.